Effect of eosin Y on the formation of local lesions and on the accumulation of callose in ‘Samsun NN’ tobacco and French bean leaves inoculated with tobacco mosaic virus

When leaf-halves of Samsun NN tobacco or bean plants were floated on a solution of 10–15 M eosin Y after inoculation with tobacco mosaic virus (TMV) and kept at 20° C, local lesion formation was markedly diminished. There was also a decrease in the size of the lesions.Depending on the temperature very strong fluorescence due to callose formation was seen around the lesions in eosin Y-treated leaf-halves of Samsun NN tobacco and bean plants. It lasted from 3–5 days after inoculation, whereas fluorescence around lesions in the water-treated control leaves disappeared within 2–3 days after inoculation.When leaf discs of Samsun tobacco, a systemic host for TMV, were floated on a solution of eosin Y after inoculation with TMV and kept at 20°C for 5 days, TMV multiplication was not prevented. Callose deposition could be detected, neither in eosin Y-treated nor in water-treated control leaves.The relation between the inhibition of local lesion formation and the accumulation of callose in eosin Y-treated leaves is discussed.Samenvatting Het is bekend dat er een verband bestaat tussen afzetting van callose en de vorming van lokale lesies bij verschillende virus-waardplantcombinaties. Tevens staat vast dat eosine ophoping van callose op zeefplaten veroorzaakt. Onderzocht werd nu of eosine Y van invloed is op de vorming van lokale lesies en tevens ophoping van callose veroorzaakt in bladeren van Samsun NN-tabak en boon, geïnoculeerd met tabaksmozaïekvirus (TMV).Het bleek dat bladhelften van tabak die, na inoculatie met TMV, bij 20°C gedreven hadden op een oplossing van 10 of 15 M eosine Y, minder en kleinere lesies hadden dan de controlehelften die op water hadden gedreven (Tabel 1; Fig. 1). Bij 25°C werden deze effecten niet waargenomen (Fig. 2). Een concentratie van 15 M veroorzaakte wat beschadiging van de bladeren (verbruining van een deel van de zijnerven).Bij bladhelften van de boon was geen beschadiging te zien bij een concentratie van 15 M. Bij bonebladeren geïnoculeerd met TMV bleek behandeling met eosine Y zowel bij 20°C als bij 25°C en zelfs bij 30°C minder en kleinere lesies tot gevolg te hebben (Tabel 2; Fig. 3 en 4).Als gezonde bladhelften van Samsun NN-planten te drijven waren gelegd op water verscheen er callose in de buurt van het wondvlak (Fig. 5). Deze ophoping van callose was sterker als de bladhelften hadden gedreven op eosine Y in concentraties van 10 of 15 M (Fig. 6). In de zijnerven, die als gevolg van de behandeling met 15 M bruin waren geworden, was eveneens een sterke fluorescentie als gevolg van de aanwezigheid van callose te zien (Fig. 7 en 8).Als bladhelften van Samsun NN-planten na inoculatie met TMV te drijven waren gelegd op oplossingen van 10 of 15 M bij een temperatuur van 20°C bleek er niet alleen een reductie in aantal en afmeting van de lesies opgetreden te zijn, maar ook sterke fluorescentie als gevolg van aanzienlijke callose-afzettingen rondom de in hun uitbreiding geremde lesies (Fig. 9). De sterke fluorescentie verdween zelfs niet 3–5 dagen na inoculatie, hoewel bij bladeren die op water hadden gedreven dit al na 2–3 dagen het geval was (Fig. 10). Hadden de bladhelften na inoculatie echter bij 25°C op eosine Y gedreven dan was de fluorescentie rondom de lesies dezelfde als in de bladhelften die op water hadden gedreven.Bij bladhelften van gezonde bonen was niet veel ophoping van wondcallose waarneembaar en evenmin stimuleerde eosine Y de vorming ervan. Wel bracht deze stof een ophoping van callose teweeg op de zeefplaten van bladnerven (Fig. 11). Eveneens was er in de met eosine Y behandelde bladhelften fluorescentie in delen van de zijnerven (Fig. 12).Bij bonebladeren die waren geïnoculeerd met TMV was een sterke afzetting van callose te zien rondom de lesies die zich niet meer uitbreidden als gevolg van behandeling met eosine Y bij temperaturen tussen 20°C en 30°C (Fig. 13). In de controlehelften die op water hadden gedreven verdween de fluorescentie binnen 2 dagen na inoculatie (Fig. 14).Hadden bladschijfjes van Samsun-tabak na inoculatie met TMV gedreven op een oplossing van eosine Y dan bleek de virusvermeerdering in deze schijfjes niet geremd te zijn (Tabel 3).We kunnen concluderen dat er een correlatie bestaat tussen de hoeveelheid afgezette callose in de bladeren en de uiteindelijke grootte van de lokale lesies. De vraag blijft echter nog wel bestaan of callose-afzetting inderdaad de verspreiding van virus naar naburige cellen verhindert.     

Sequence diversity in the coat protein and 3″-untranslated region of Chinese yam necrotic mosaic virus RNA

Sixteen isolates of Chinese yam necrotic mosaic virus (ChYNMV) were collected from nine sites in Japan and one site in Korea, and 1098 nucleotides of the 3-terminal of the genome were sequenced. Identity of the coat protein gene was 95.5%–99.7% among the isolates. Substitution in the deduced amino acids of the coat protein ranged from 0 to 7, mainly in the N-terminal region. The 3-untranslated region consisted of 231 nucleotides, which had 96.5%–100% nucleotide identity among the isolates. Sequence diversity was considerably less in ChYNMV than in Yam mosaic virus or Japanese yam mosaic virus.     

Effect of the disodium salt of cyclopentene β,β′-triketone on the development of infections induced by TMV in the leaves of hypersensitive and susceptible tobacco plants

Journal of Plant Diseases and Protection - We show here that the disodium salt of 2-acetyl-5-chloro-4- hydroxycarbonylmethylthiocyclopent-4-ene-1,3-dione (salt 2) inhibits tobacco mosaic virus...     

Does agricultural use of azole fungicides contribute to resistance in the human pathogen Aspergillus fumigatus?

Azole resistance in human fungal pathogens has increased over the past twenty years, especially in immunocompromised patients. Similarities between medical and agricultural azoles, and extensive azole (14α‐demethylase inhibitor, DMI) use in crop protection, prompted speculation that resistance in patients with aspergillosis originated in the environment. Aspergillus species, and especially Aspergillus fumigatus, are the largest cause of patient deaths from fungi. Azole levels in soils following crop spraying, and differences in sensitivity between medical and agricultural azoles (DMIs), indicate weaker selection in cropping systems than in patients receiving azole therapy. Most fungi have just one CYP51 paralogue (isozyme CYP51B), but in Aspergillus sp. mutations conferring azole resistance are largely confined to a second paralogue, CYP51A. Binding within the active centre is similar for medical and agricultural azoles but differences elsewhere between the two paralogues may ensure selection depends on the DMI used on crops. Two imidazoles, imazalil and prochloraz, have been widely used since the early 1970s, yet unlike triazoles they have not been linked to resistance in patients. Evidence that DMIs are the origin, or increase the frequency, of azole resistance in human fungal pathogens is lacking. Limiting DMI use would have serious impacts on disease control in many crops, and remove key tools in anti‐resistance strategies. © 2017 Society of Chemical Industry     

Expression of the yeast Δ-9 desaturase gene in tomato enhances its resistance to powdery mildew

Tomato plants (Lycopersicon esculentumMill.) transformed with the yeast Δ-9 desaturase gene were found to display enhanced resistance to powdery mildew (Erysiphe polygoniDC. edmund. Salm.) and to possess higher levels of 16:1, 16:2 and 18:1 fatty acids. Lipid peroxides also increased. The improved resistance of the transgenic plants was apparently associated with the increases in 16:1 and 16:2 fatty acids.     

Do some IPM concepts contribute to the development of fungicide resistance? Lessons learned from the apple scab pathosystem in the United States

One goal of integrated pest management (IPM) as it is currently practiced is an overall reduction in fungicide use in the management of plant disease. Repeated and long‐term success of the early broad‐spectrum fungicides led to optimism about the capabilities of fungicides, but to an underestimation of the risk of fungicide resistance within agriculture. In 1913, Paul Ehrlich recognized that it was best to ‘hit hard and hit early’ to prevent microbes from evolving resistance to treatment. This tenet conflicts with the fungicide reduction strategies that have been widely promoted over the past 40 years as integral to IPM. The authors hypothesize that the approaches used to implement IPM have contributed to fungicide resistance problems and may still be driving that process in apple scab management and in IPM requests for proposals. This paper also proposes that IPM as it is currently practiced for plant diseases of perennial systems has been based on the wrong model, and that conceptual shifts in thinking are needed to address the problem of fungicide resistance. © 2013 Society of Chemical Industry     

Interactions in the Brassica napus–Pyrenopeziza brassicae pathosystem and sources of resistance to P. brassicae (light leaf spot)

Pyrenopeziza brassicae, cause of light leaf spot (LLS), is an important pathogen of oilseed rape and vegetable brassicas and has a wide geographic distribution. Exploitation of host resistance remains the most sustainable and economically viable solution for disease management. This study evaluated 18 oilseed rape cultivars or breeding lines for host resistance against P. brassicae in glasshouse experiments. Selected cultivars/lines were inoculated with eight single-spore isolates of the pathogen obtained from three different regions in England. Analysis of P. brassicae infection-related changes on host plants identified leaf deformation as a characteristic feature associated with P. brassicae infection, this showed poor correlation to LLS severity measured as the amount of pathogen sporulation on infected plants. Resistant host phenotypes were identified by limitation of P. brassicae sporulation, with or without the presence of a necrotic response (black flecking phenotype). Investigation of this pathosystem revealed significant differences between cultivars/lines, between isolates, and significant cultivar/line-by-isolate interactions. In total, 37 resistant and 16 moderately resistant interactions were identified from 144 cultivar/line-by-isolate interactions using statistical methods. Most of the resistant/moderately resistant interactions identified in this study appeared to be nonspecific towards the isolates tested. Our results suggested the presence of isolate-specific resistant interactions for some cultivars. Several sources of resistance have been identified that are valuable for oilseed rape breeding programmes.     

Multiple facets of response to fungicides – the influence of azole treatment on expression of key mycotoxin biosynthetic genes and candidate resistance factors in the control of resistant <Emphasis Type="Italic">Fusarium</Emphasis> strains

The spread of fungicide resistant and/or tolerant phytopathogenic fungi is an important factor affecting crop protection. However, the mechanisms of fungal response to fungicide application are not entirely characterised. In particular, the contribution of previously known resistance factors and the final influence of fungicide treatments on metabolism of surviving mycelia (e.g. mycotoxin increased release and biosynthesis potentially causing contamination of the crops) merit investigation, in order to improve future molecular diagnostics of fungicide resistant strains. The performed experiments have shown distinct expression changes for homologs of a known fungicide resistance factor Flr1 (yeast; DHA1 family of major facilitator superfamily transporters) after azole application in cultured fusaria. Two distantly related homologs of that gene were selected, based on the unsupervised clustering and phylogenetic analysis of transporter sequences. One of these (FGSG_02865), was found to occur across the Fusarium sambucinum complex (F. graminearum, F. culmorum, F. cerealis) and was upregulated starting 24 h after fungicide treatments. This delayed response may point to possible involvement of DHA1 antiporters in a generalised response to stress resulting from fungicide treatment. Additional expression profiling was conducted for the mycotoxin biosynthetic genes (trichothecene and zearalenone gene clusters) in strains of Fusarium sambucinum complex cereal pathogens. The expression changes, when subjected to treatment with the fungicides (flusilazole, carbendazim), show that even an effective treatment (in this study, the benzimidazole fungicide carbendazim) applied to the grown mycelium, can result in enhanced activation of mycotoxin biosynthetic genes in fungal cells which survive the treatment. Our results suggest that increased mycotoxin contamination can be strongly influenced not only by the amount or the type of antifungal compound, but also the timing of fungicide exposition (stage of infection).     

Susceptibility to neonicotinoids and risk of resistance development in the brown planthopper, Nilaparvata lugens (Stål) (Homoptera: Delphacidae)

BACKGROUND: In recent years, outbreaks of the brown planthopper, Nilaparvata lugens (Stål), have occurred more frequently in China. The objective of this study was to determine the susceptibility of N. lugens to neonicotinoids and other insecticides in major rice production areas in China. RESULTS: Results indicated that substantial variations in the susceptibility to different insecticides existed in N. lugens. Field populations had developed variable resistance levels to neonicotinoids, with a high resistance level to imidacloprid (RR: 135.3–301.3‐fold), a medium resistance level to imidaclothiz (RR: 35–41.2‐fold), a low resistance level to thiamethoxam (up to 9.9‐fold) and no resistance to dinotefuran, nitenpyram and thiacloprid (RR < 3‐fold). Further examinations indicated that a field population had developed medium resistance level to fipronil (up to 10.5‐fold), and some field populations had evolved a low resistance level to buprofezin. In addition, N. lugens had been able to develop 1424‐fold resistance to imidacloprid in the laboratory after the insect was selected with imidacloprid for 26 generations. CONCLUSION: Long‐term use of imidacloprid in a wide range of rice‐growing areas might be associated with high levels of resistance in N. lugens. Therefore, insecticide resistance management strategies must be developed to prevent further increase in resistance. Copyright © 2008 Society of Chemical Industry     

Identifying potential novel resistance to the foliar disease ‘Scald’ (Rhynchosporium commune) in a population of Scottish Bere barley landrace (Hordeum vulgare L.)

Journal of Plant Diseases and Protection - Barley ‘Scald’ is an economically damaging fungal disease that is a global problem, causing significant yield and economical losses in the UK...     

Analysis of the relationship between parameters of resistance to <Emphasis Type="Italic">Fusarium</Emphasis> head blight and <Emphasis Type="Italic">in vitro</Emphasis> tolerance to deoxynivalenol of the winter wheat cultivar WEK0609 <Superscript>®</Superscript>

Fusarium head blight (FHB) symptom development, relative spikelet weight (RSW), fungal DNA (FDNA) and deoxynivalenol (DON) content of grain was assessed in the FHB resistant winter wheat cv. WEK0609 and the FHB susceptible cv. Hobbit sib, and among doubled haploid progeny lines (DHLs) developed from a cross between these cultivars. In addition, the relationship between FHB resistance traits and germination on DON-containing medium (in vitro DON tolerance (IVDT)) was also investigated to assess the possibility of using this test as in vitro method of screening for FHB resistance in this cultivar. Analysis indicated that WEK0609 resistance significantly reduced symptom development, yield loss and the FDNA and DON content of grain relative to Hobbit sib. Although both the DON and FDNA content were greater in susceptible than in resistant progeny lines, the ratio of DON to FDNA decreased with increasing susceptibility. The resistance derived from WEK0609 appears to have a greater effect on colonisation of the grain by the fungus than on the accumulation of DON within the grain. In vitro tolerance to DON does not appear to relate to FHB resistance in WEK0609 and thus does not provide a means of selecting for FHB resistance derived from this cultivar.     

Identification of a novel point mutation in the <Emphasis Type="Italic">β</Emphasis>-tubulin gene of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> and detection of benzimidazole resistance by a diagnostic PCR-RFLP assay

The molecular basis of resistance to benzimidazole fungicides with laboratory and field mutant isolates of Botrytis cinerea was investigated. After chemical mutagenesis with N-methyl-N-nitrosogouanidine (NMNG) two different benzimidazole-resistant phenotypes were isolated on media containing carbendazim or a mixture of carbendazim and diethofencarb. The mutant isolates from the fungicide-mixture-containing medium were moderately resistant to carbendazim with wild-type tolerance to diethofencarb while mutant isolates from carbendazim-containing medium were highly resistant to carbendazim but sensitive to diethofencarb. The studied field isolates were highly resistant to benzimidazoles and sensitive to diethofencarb. Study of fitness characteristics of benzimidazole highly-resistant isolates showed that the resistance mutation(s) had no apparent effect on fitness-determining parameters. Contrary to this, the moderately benzimidazole-resistant strains, with no increased diethofencarb sensitivity, had a significant reduction in certain ecological fitness-determining characteristics. Analysis of the sequence of the β-tubulin gene revealed two amino acid replacements in the highly benzimidazole-resistant mutants compared to that of the wild-type parent strain. One was the glutamic acid (GAG) to alanine (GCG) change at position 198 (E198A), identified in both laboratory and field highly benzimidazole-resistant isolates, a mutation previously implicated in benzimidazole resistance. The second was a novel benzimidazole resistance mutation of glutamic acid (GAG) to glycine (GGG) substitution at the same position 198 (E198G), identified in a highly benzimidazole-resistant laboratory mutant strain. Molecular analysis of the moderately benzimidazole-resistant strains revealed no mutations at the β-tubulin gene. A novel diagnostic PCR-RFLP assay utilising a BsaI restriction site present in the benzimidazole-sensitive (E198) but absent in both resistant genotypes (E198G and E198A) was developed for the detection of both amino acid replacements at the β-tubulin gene.     

Suppression of mRNAs for lipoxygenase (LOX), allene oxide synthase (AOS), allene oxide cyclase (AOC) and 12-oxo-phytodienoic acid reductase (OPR) in pea reduces sensitivity to the phytotoxin coronatine and disease development by Mycosphaerella pinodes

Using a recently developed model pathosystem involving Medicago truncatula and Mycosphaerella pinodes, causal agent of Mycosphaerella blight on pea to understand host molecular response to a fungal suppressor, we applied the suppressor to leaves of M. truncatula and identified 151 nonredundant cDNA fragments as newly expressed genes. These included genes encoding lipoxygenase (LOX) and enoyl-CoA hydratase, which are presumably involved in jasmonic acid (JA) synthesis. Potential genes encoding plastidic enzymes, including allene oxide synthase (AOS) and allene oxide cyclase (AOC), and other peroxisomal enzymes involved in β-oxidation were predicted from the Medicago Gene Index EST database and tested for altered expression by semiquantitative RT-PCR. The coordinated expression of genes encoding both plastidic and peroxisomal enzymes showed that the suppressor likely conditions certain cellular process(es) through the JA synthesis in M. truncatula. To explore the role of JA or JA-regulated cellular process(es) in conditioning susceptibility, we used an Apple latent spherical virus (ALSV)-based virus-induced gene silencing (VIGS) technology to silence pea genes including LOX, AOS, AOC and 12-oxo-phytodienoic acid reductase (OPR). In LOX-, AOS-, AOC- or OPR-silenced pea plants, disease development induced by M. pinodes was remarkably reduced. Similarly, silencing of mRNA for LOX, AOS, AOC or OPR reduced the sensitivity to a phytotoxin, coronatine, which is believed to act through a JA-dependent process. On the basis of these results, it is conceivable that M. pinodes has evolved a strategy to condition susceptibility by manipulating the physiology of host cells, in particular JA-regulated cellular process(es), to promote disease development in pea.     

Response to the letter on “Climatic distribution of citrus black spot caused by <Emphasis Type="Italic">Phyllosticta citricarpa.</Emphasis> A historical analysis of disease spread in South Africa” by Fourie et al. (2017)

In a previous study, Martínez-Minaya et al. (European Journal of Plant Pathology 143, 69–83, 2015) performed an analysis of climate-based distribution of citrus black spot (CBS) in South Africa. It was found that CBS was initially confined to humid areas with summer rainfall, but later spread to arid steppe and even desert climates. A strong spatial autocorrelation of CBS distribution was found. Fourie et al. (European Journal of Plant Pathology, 2017) take a critical view of our study, but without presenting any analysis of results to refute our findings. Furthermore, Fourie et al. (European Journal of Plant Pathology, 2017) appear to have misunderstood our work, since many of their criticisms relate to the potential distribution of CBS in Europe, which is beyond the scope of our original study. Fourie et al. (European Journal of Plant Pathology, 2017) highlight the limitations of climate classifications in species distribution modelling. However, this was made explicit in our study, indicating that it was a preparatory work and further advanced modelling studies, including spatial effects, will be needed. Fourie et al. (European Journal of Plant Pathology, 2017) incorrectly assume that we used all of South Africa as the background in the spatial autocorrelation analysis. However, only citrus areas were used and a strong spatial autocorrelation was detected at all distances evaluated. Contrary to what Fourie et al. (European Journal of Plant Pathology, 2017) suggest, similar climate distributions of CBS were obtained at 5′ and 30′ resolution, and also with the national land-cover map of South Africa. The figure comparison presented by Fourie et al. (European Journal of Plant Pathology, 2017) appears to ignore the fact that the maps we used were grid cells of 10 × 10 km and not the line polygons they suggest. Therefore, we consider the conclusions from the Martínez-Minaya et al. (European Journal of Plant Pathology 143, 69–83, 2015) remain entirely valid.